Chromatography columns have been extensively developed and are used routinely in both analytical and preparative chromatography. As is well known, the separation in a chromatography column of a sample (also termed an analyte or solute) comprising a mixture of components is achieved by dissolving the sample in an eluant to form a fluid mobile phase and passing the mobile phase through a stationary phase typically packed within a tubular column, thereby causing the sample to separate into its components due to the differences in the partitioning between the mobile and stationary phases of the different components (i.e. the components have different partition coefficients). The present invention relates to such columns.
The eluant fluid is most commonly a liquid but may be another fluid such as a supercritical fluid (SCF). The present invention relates to columns used with liquid or SCF mobile phases. The term fluid herein thus refers to liquid or SCF. The present invention, for the avoidance of doubt, does not relate to gas chromatography.
In column chromatography the stationary phase is typically in the form of a bed of packed particles or a porous monolithic block within a column. The present invention is applicable to packed columns but it is not limited to only packed columns. Often the columns comprise reusable columns with disposable cartridges, both of which are usually cylindrical (typically circular cross section). The present invention may be used with cylindrical columns (most preferably circular cylindrical columns) or non-cylindrical columns. That is, the wall of the column may have numerous cross-sectional profiles but most preferably has a circular profile in its transverse cross section.
The mobile phase is passed through the column and the mobile phase leaving the column (termed eluate) is detected as a function of time and/or collected as fractions. The detected signal variation with time, i.e. the chromatogram, indicates the presence of different components within the mixture. The degree of separation of the different components depends upon the separation efficiency or resolution of the column. The resolution of the column depends upon many factors. Such factors include the nature of the mobile and stationary phases, which have been extensively studied and developed.
In the technique of reaction chromatography the chemical identities of the sample components are changed by a chemical reaction occurring between sample introduction and sample detection. The reaction can take place upstream of the column such that the chemical identities of the individual components passing through the column differ from those of the original sample, or downstream of the column, i.e. between the column and the detector, such that the original (i.e. unreacted) sample components are separated in the column but their identities are changed prior to being detected by the detector. The reaction can also take place between two columns. For example, a first column may separate enantiomers, and a chemical reaction is targeted to, perhaps just one, or both, enantiomeric species. The reaction occurs, and the second column then separates subsequent products from the reaction.
Applications of reaction chromatography can be found, for example, in the biosciences, particularly in the area of proteomics, as well as in the pharmaceutical and environmental industries. In such applications, analysts will typically perform some type of post-column modification of the samples. For example, post-column derivatisation of the samples is performed to allow detection and analysis of the sample by means of a suitable detector employing, for example fluorescence detection, chemiluminescence detection or a decolorisation reaction, but not limited to these types of detections. The detection method based on the reaction may be more sensitive, compared to detection of the unreacted sample but more importantly the detection methods provide greater detection specificity, i.e. the reaction and associated detection method are specific to certain components of the sample and not others.
In proteomics, often one or two proteins are desired from an entire proteome for further characterization. Analysis of the target proteins has been achieved via two principle processes: top-down or bottom-up proteomics. The former process involves the characterization of intact proteins, whilst the latter involves the use of digestive enzymes followed by the characterization of fragments. Both approaches have used High Performance Liquid Chromatography (HPLC) and a protein modification has been performed as a separate step either before or after the chromatography column.
As each step of a process typically requires more time, labour and cost, the elimination or simplification of any step is economically desirable and may bring about enhancements in sample throughput and processing.